Maize genomic marker set

ABSTRACT

Maize markers useful for genotyping and association studies, e.g. association with oil content QTLs in populations derived from the Illinois High Oil and Illinois Low Oil maize lines. Primers and hybridization probes for Taqman™ assays are provided for 488 SNP markers in 484 loci.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 120 to U.S. application Ser. No. 10/806,705, filed Mar. 22, 2004, application Ser. No. 10/613,520, filed Jul. 2, 2003, application Ser. No. 10/389,566, filed Mar. 14, 2003 which claims priority under 35 U.S.C. 119(e) to U.S. Provisional Applications 60/365,301 filed Mar. 15, 2002, 60/391,786 filed Jun. 25, 2002 and 60/392,018 filed Jun. 26, 2002, all of which are incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

Two copies and a computer readable form of the sequence listing, i.e. labeled “Copy 1 of Sequence Listing”, “Copy 2 of Sequence Listing” and “Computer Readable Form of Sequence Listing”, each on a separate CD-ROM containing the file named “52900H.ST25.txt”, which is 788 kb (measured in MS-Windows) and was created on Jul. 22, 2004, are herein incorporated by reference.

INCORPORATION OF TABLES

Two copies of Table 1, labeled as “Copy 1 of Table 1” and “Copy 2 of Table 1”, each on a separate CD-ROM containing the file named “Table 1.txt”, which is 145 kb (measured in MS-Windows) and was created on Jul. 22, 2004, are herein incorporated by reference.

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant molecular biology, plant genetics and plant breeding. More specifically disclosed are maize genetic markers, i.e. polymorphic DNA, which are useful for discovery and isolation of genes, marker trait association, discovery of QTLs and molecular breeding.

BACKGROUND OF THE INVENTION

Maize, Zea mays L., is one of the major crops grown worldwide as a primary source for animal feed, human food and industrial purposes. Maize plants with improved agronomic traits and maize seed with improved quality traits are desirable for the farmer, processor and consumer of maize and maize derived products. The ability to breed or develop transgenic plants with improved traits depends in part on identification of genes or QTLs associated with a trait. The unique maize sequences disclosed herein are useful as mapping tools to assist in plant breeding, in gene and QTL discovery, as markers in marker trait association and molecular breeding.

Polymorphisms are useful as genetic markers for genotyping applications in the agriculture field, e.g., in plant genetic studies and commercial breeding. See for instance U.S. Pat. Nos. 5,385,835; 5,492,547 and 5,981,832, the disclosures of all of which are incorporated herein by reference. The highly conserved nature of DNA combined with the rare occurrences of stable polymorphisms provide genetic markers that are both predictable and discerning of different genotypes. Among the classes of existing genetic markers are a variety of polymorphisms indicating genetic variation including restriction-fragment-length polymorphisms (RFLPs), amplified fragment-length polymorphisms (AFLPs), simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), and insertion/deletion polymorphisms (Indels). Because the number of genetic markers for a plant species is limited, the discovery of additional genetic markers associated with a trait will facilitate genotyping applications including marker-trait association studies, gene mapping, gene discovery, marker-assisted selection, and marker-assisted breeding. Evolving technologies make certain genetic markers more amenable for rapid, large scale use. For instance, technologies for SNP detection indicate that SNPs may be preferred genetic markers.

SUMMARY OF THE INVENTION

This invention provides maize polymorphic markers, more specifically SNP and Indel markers located in 484 polymorphic maize genomic DNA loci having DNA sequence of SEQ ID NO:1 through SEQ ID NO:484. Such markers are useful for discovery and isolation of genes, marker trait association, discovery of QTLs and molecular breeding. This invention also provides primers and probes useful in genotyping with the 488 specific markers within the 484 polymorphic loci. This invention also provides methods of using markers in the 484 polymorphic loci for genotyping maize, e.g. in identifying genes and QTLs, molecular breeding, mapping DNA clones, and the like.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following definitions are used to described the markers and their uses.

An “allele” means an alternative sequence at a particular locus; the length of an allele can be as small as 1 nucleotide base but is typically larger. Allelic sequence can be amino acid sequence or nucleic acid sequence.

A “locus” is a short sequence that is usually unique and usually found at one particular location by a point of reference, e.g., a short DNA sequence that is a gene, or part of a gene or intergenic region. A locus of this invention can be a unique PCR product. The loci of this invention are polymorphic between certain individuals.

“Genotype” means the specification of an allelic composition at one or more loci within an individual organism. In the case of diploid organisms, there are two alleles at each locus; a diploid genotype is said to be homozygous when the alleles are the same, and heterozygous when the alleles are different.

“Phenotype” means the detectable characteristics of a cell or organism that are a manifestation of gene expression.

“Marker” means a polymorphic sequence. A “polymorphism” is a variation among individuals in sequence, particularly in DNA sequence. Useful polymorphisms include a single nucleotide polymorphisms (SNPs) and insertions or deletions in DNA sequence (Indels).

“Marker assay” means a method for detecting a polymorphism at a particular locus using a particular method, e.g., phenotype (such as seed color, flower color, or other visually detectable trait), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allelic specific oligonucleotide hybridization (ASO), RAPID, etc.

Preferred marker assays include single base extension as disclosed in U.S. Pat. No. 6,013,431 and allelic discrimination where endonuclease activity releases a reporter dye from a hybridization probe as disclosed in U.S. Pat. No. 5,538,848, the disclosures of both of which are incorporated herein by reference.

“Linkage” refers to relative frequency at which types of gametes are produced in a cross. For example, if locus A has alleles “A” or “a” and locus B has alleles “B” or “b,” a cross between parent I with AABB and parent II with aabb will produce four possible gametes where the haploid genotypes are segregated into AB, Ab, aB and ab. The null expectation is that there will be independent and equal segregation into each of the four possible genotypes, i.e., with no linkage, ¼ of the gametes will be of each genotype. Segregation of gametes into a genotypes differing from ¼ are attributed to linkage. Two loci are said to be “genetically linked” when they show this deviation from the expected equal frequency of ¼.

“Linkage disequilibrium” is defined in the context of the relative frequency of gamete types in a population of many individuals in a single generation. If the frequency of allele A is p, a is p′, B is q and b is q′, then the expected frequency (with no linkage disequilibrium) of genotype AB is pq, Ab is pq′, aB is p′q and ab is p′q′. Any deviation from the expected frequency is called linkage disequilibrium.

“Quantitative Trait Locus (QTL)” means a locus that controls to some degree numerically representable traits that are usually continuously distributed.

“Haplotype” means the genotype for multiple loci or genetic markers in a haploid gamete. Generally these loci or markers reside within a relatively small and defined region of a chromosome. A preferred haplotype comprises the 10 cM region or the 5 cM region or the 2 cM region surrounding an informative marker having a significant association with oil.

“Hybridizing” means the capacity of two nucleic acid molecules or fragments thereof to to form anti-parallel, double-stranded nucleotide structure. The nucleic acid molecules of this invention are capable of hybridizing to other nucleic acid molecules under certain circumstances. A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if the molecules exhibit “complete complementarity,” i.e., each nucleotide in one sequence is complementary to its base pairing partner nucleotide in another sequence. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Nucleic acid molecules that hybridize to other nucleic acid molecules, e.g., at least under low stringency conditions are said to be “hybridizable cognates” of the other nucleic acid molecules. Conventional stringency conditions are described by Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and by Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985), each of which is incorporated herein by reference. Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. Thus, in order for a nucleic acid molecule to serve as a primer or probe, it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed. Appropriate stringency conditions that promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated herein by reference. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.

“Sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc. Burlington, Mass.). Polynucleotides of the present invention that are variants of the polynucleotides provided herein will generally demonstrate significant identity with the polynucleotides provided herein. Of particular interest are polynucleotide homologs having at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, and more preferably even greater, such as 98% or 99% sequence identity with polynucleotide sequences described herein.

“Purified” refers to a nucleic acid molecule or polypeptide separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule may be greater than 60% free or 75% free or 90% free or 95% free from the other molecules (exclusive of solvent) present in the natural mixture. The terms “isolated and purified” and “substantially purified” are not intended to encompass molecules present in their native state.

Characteristics of Maize Markers

The maize loci of this invention comprise a DNA sequence that comprises at least 20 consecutive nucleotides and includes or is adjacent to one or more polymorphisms identified in Table 1. Such maize loci have a nucleic acid sequence having at least 90% sequence identity or at least 95% or for some alleles at least 98% and in many cases at least 99% sequence identity, to the sequence of the same number of nucleotides in either strand of a segment of maize DNA that includes or is adjacent to the polymorphism. The nucleotide sequence of one strand of such a segment of maize DNA may be found in a polymorphic locus with a sequence in the group consisting of SEQ ID NO:1 through SEQ ID NO:186. It is understood by the very nature of polymorphisms that for at least some alleles there will be no identity to the polymorphism, per se. Thus, sequence identity can be determined for sequence that is exclusive of the polymorphism sequence. The polymorphisms in each locus are identified more particularly in Table 1.

For many genotyping applications it is useful to employ as markers polymorphisms from more than one locus. Thus, aspects of the invention use a collection of different loci. The number of loci in such a collection can vary but will be a finite number, e.g., as few as 2 or 5 or 10 or 25 loci or more, for instance up to 40 or 75 or 100 or more loci.

Another aspect of the invention provides nucleic acid molecules that are capable of hybridizing to the polymorphic maize loci of this invention, e.g. PCR primers and hybridization probes. In certain embodiments of the invention, e.g., which provide PCR primers, such molecules comprise at least 15 nucleotide bases. Molecules useful as primers can hybridize under high stringency conditions to one of the strands of a segment of DNA in a polymorphic locus of this invention. Primers for amplifying DNA are provided in pairs, i.e., a forward primer and a reverse primer. One primer will be complementary to one strand of DNA in the locus and the other primer will be complementary to the other strand of DNA in the locus, i.e., the sequence of a primer is at least 90% or at least 95% identical to a sequence of the same number of nucleotides in one of the strands. It is understood that such primers can hybridize to a sequence in the locus that is distant from the polymorphism, e.g., at least 5, 10, 20, 50 or up to about 100 nucleotide bases away from the polymorphism. Design of a primer of this invention will depend on factors well known in the art, e.g., avoidance of repetitive sequence.

Another aspect of the nucleic acid molecules of this invention are hybridization probes for polymorphism assays. In one aspect of the invention such probes are oligonucleotides comprising at least 12 nucleotide bases and a detectable label. The purpose of such a molecule is to hybridize, e.g., under high stringency conditions, to one strand of DNA in a segment of nucleotide bases that includes or is adjacent to the polymorphism of interest in an amplified part of a polymorphic locus. Such oligonucleotides are at least 90% or at least 95% identical to the sequence of a segment of the same number of nucleotides in one strand of maize DNA in a polymorphic locus. The detectable label can be a radioactive element or a dye. In preferred aspects of the invention, the hybridization probe further comprises a fluorescent label and a quencher, e.g., for use in hybridization probe assays of the type known as Taqman assays, available from Applied Biosystems of Foster City, Calif.

For assays where the molecule is designed to hybridize adjacent to a polymorphism that is detected by single base extension, e.g., of a labeled dideoxynucleotide, such molecules can comprise at least 15 or at least 16 or 17 nucleotide bases in a sequence that is at least 90% or at least 95% identical to a sequence of the same number of consecutive nucleotides in either strand of a segment of polymorphic maize DNA. Oligonucleotides for single base extension assays are available from Orchid Bioystems.

Such primer and probe molecules are generally provided in groups of two primers and one or more probes for use in genotyping assays. Moreover, it is often desirable to conduct a plurality of genotyping assays for a plurality of polymorphisms. Thus, this invention also provides collections of nucleic acid molecules, e.g., in sets that characterize a plurality of polymorphisms.

Detecting Polymorphisms

Polymorphisms in DNA sequences can be detected by a variety of effective methods well known in the art including those methods disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863 by hybridization to allele-specific oligonucleotides; in U.S. Pat. Nos. 5,468,613 and 5,800,944 by probe ligation; in U.S. Pat. No. 5,616,464 by probe linking; and in U.S. Pat. Nos. 6,004,744; 6,013,431; 5,595,890; 5,762,876; and 5,945,283 by labeled base extension, all of which are incorporated herein by reference.

In another preferred method for detecting polymorphisms, SNPs and Indels can be detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930; and 6,030,787 in which an oligonucleotide probe having a 5′fluorescent reporter dye and a 3′quencher dye covalently linked to the 5′ and 3′ ends of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in the suppression of the reporter fluorescence, e.g., by Forster-type energy transfer. During PCR forward and reverse primers hybridize to a specific sequence of the target DNA flanking a polymorphism. The hybridization probe hybridizes to polymorphism-containing sequence within the amplified PCR product. In the subsequent PCR cycle DNA polymerase with 5′→3′ exonuclease activity cleaves the probe and separates the reporter dye from the quencher dye resulting in increased fluorescence of the reporter. A useful assay is available from AB Biosystems as the Taqman® assay, which employs four synthetic oligonucleotides in a single reaction that concurrently amplifies the maize genomic DNA, discriminates between the alleles present, and directly provides a signal for discrimination and detection. Two of the four oligonucleotides serve as PCR primers and generate a PCR product encompassing the polymorphism to be detected. Two others are allele-specific fluorescence-resonance-energy-transfer (FRET) probes. FRET probes incorporate a fluorophore and a quencher molecule in close proximity so that the fluorescence of the fluorophore is quenched. The signal from a FRET probe is generated by degradation of the FRET oligonucleotide, so that the fluorophore is released from proximity to the quencher, and is thus able to emit light when excited at an appropriate wavelength. In the assay, two FRET probes bearing different fluorescent reporter dyes are used, where a unique dye is incorporated into an oligonucleotide that can anneal with high specificity to only one of the two alleles. Useful reporter dyes include 6-carboxy-4,7,2′,7′-tetrachlorofluorecein (TET), VIC (a dye from Applied Biosystems Foster City, Calif.), and 6-carboxyfluorescein phosphoramidite (FAM). A useful quencher is 6-carboxy-N,N,N′,N′-tetramethylrhodamine (TAMRA). Additionally, the 3′end of each FRET probe is chemically blocked so that it cannot act as a PCR primer. During the assay, maize genomic DNA is added to a buffer containing the two PCR primers and two FRET probes. Also present is a third fluorophore used as a passive reference, e.g., rhodamine X (ROX), to aid in later normalization of the relevant fluorescence values (correcting for volumetric errors in reaction assembly). Amplification of the genomic DNA is initiated. During each cycle of the PCR, the FRET probes anneal in an allele-specific manner to the template DNA molecules. Annealed (but not non-annealed) FRET probes are degraded by TAQ DNA polymerase as the enzyme encounters the 5′ end of the annealed probe, thus releasing the fluorophore from proximity to its quencher. Following the PCR reaction, the fluorescence of each of the two fluorescers, as well as that of the passive reference, is determined fluorometrically. The normalized intensity of fluorescence for each of the two dyes will be proportional to the amounts of each allele initially present in the sample, and thus the genotype of the sample can be inferred.

To design primers and probes for the assay the locus sequence is first masked to prevent design of any of the three primers to sites that match known maize repetitive elements (e.g., transposons) or arc of very low sequence complexity (di- or tri-nucleotide repeat sequences). Design of primers to such repetitive elements will result in assays of low specificity, through amplification of multiple loci or annealing of the FRET probes to multiple sites.

PCR primers are designed (a) to have a length in the size range of 18 to 25 bases and matching sequences in the polymorphic locus, (b) to have a calculated melting temperature in the range of 57° C. to 60° C., e.g., corresponding to an optimal PCR annealing temperature of 52° C. to 55° C., (c) to produce a product that includes the polymorphic site and has a length in the size range of 75 to 250 base pairs. The PCR primers are preferably located on the locus so that the polymorphic site is at least one base away from the 3′ end of each PCR primer. The PCR primers must not contain regions that are extensively self- or inter-complementary.

FRET probes are designed to span the sequence of the polymorphic site, preferably with the polymorphism located in the 3′ most 2/3 of the oligonucleotide. In the preferred embodiment, the FRET probes will have incorporated at their 3′end a chemical moiety that, when the probe is annealed to the template DNA, binds to the minor groove of the DNA, thus enhancing the stability of the probe-template complex. The probes should have a length in the range of 12 to 17 bases and, with the 3′MGB, have a calculated melting temperature of 5° C. to 7° C. above that of the PCR primers. Probe design is disclosed in U.S. Pat. Nos. 5,538,848; 6,084,102; and 6,127,121.

Use of Polymorphisms to Establish Marker/Trait Associations

The polymorphisms in the loci of this invention can be used in marker/trait associations that are inferred from statistical analysis of genotypes and phenotypes of the members of a population. These members may be individual organisms, e.g., maize, families of closely related individuals, inbred lines, dihaploids or other groups of closely related individuals. Such maize groups are referred to as “lines”, indicating line of descent. The population may be descended from a single cross between two individuals or two lines (e.g., a mapping population) or it may consist of individuals with many lines of descent. Each individual or line is characterized by a single or average trait phenotype and by the genotypes at one or more marker loci.

Several types of statistical analysis can be used to infer marker/trait association from the phenotype/genotype data, but a basic idea is to detect markers, i.e., polymorphisms, for which alternative genotypes have significantly different average phenotypes. For example, if a given marker locus A has three alternative genotypes (AA, Aa and aa), and if those three classes of individuals have significantly different phenotypes, then one infers that locus A is associated with the trait. The significance of differences in phenotype may be tested by several types of standard statistical tests such as linear regression of marker genotypes on phenotype or analysis of variance (ANOVA). Commercially available, statistical software packages commonly used to do this type of analysis include SAS Enterprise Miner (SAS Institute Inc., Cary, N.C.) and Splus (Insightful Corporation. Cambridge, Mass.).

Often the goal of an association study is not simply to detect marker/trait associations, but to estimate the location of genes affecting the trait directly (i.e., QTLs) relative to the marker locations. In a simple approach to this goal, one makes a comparison among marker loci of the magnitude of difference among alternative genotypes or the level of significance of that difference. Trait genes are inferred to be located nearest the marker(s) that have the greatest associated genotypic difference. In a more complex analysis, such as interval mapping (Lander and Botstein, Genetics 121:185-199, 1989), each of many positions along the genetic map (say at 1 cM intervals) is tested for the likelihood that a QTL is located at that position. The genotype/phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio). When the LOD score exceeds a critical threshold value, there is significant evidence for the location of a QTL at that position on the genetic map (which will fall between two particular marker loci).

1. Linkage Disequilibrium Mapping and Association Studies

Another approach to determining trait gene location is to analyze trait-marker associations in a population within which individuals differ at both trait and marker loci. Certain marker alleles may be associated with certain trait locus alleles in this population due to population genetic process such as the unique origin of mutations, founder events, random drill and population structure. This association is referred to as linkage disequilibrium. In linkage disequilibrium mapping, one compares the trait values of individuals with different genotypes at a marker locus. Typically, a significant trait difference indicates close proximity between marker locus and one or more trait loci. If the marker density is appropriately high and the linkage disequilibrium occurs only between very closely linked sites on a chromosome, the location of trait loci can be very precise.

A specific type of linkage disequilibrium mapping is known as association studies. This approach makes use of markers within candidate genes, which are genes that are thought to be functionally involved in development of the trait because of information such as biochemistry, physiology, transcriptional profiling and reverse genetic experiments in model organisms. In association studies, markers within candidate genes are tested for association with trait variation. If linkage disequilibrium in the study population is restricted to very closely linked sites (i.e., within a gene or between adjacent genes), a positive association provides nearly conclusive evidence that the candidate gene is a trait gene.

2. Positional Cloning and Transgenic Applications

Traditional linkage mapping typically localizes a trait gene to an interval between two genetic markers (referred to as flanking markers). When this interval is relatively small (say less than 1 Mb), it becomes feasible to precisely identify the trait gene by a positional cloning procedure. A high marker density is required to narrow down the interval length sufficiently. This procedure requires a library of large insert genomic clones (such as a BAC library), where the inserts are pieces (usually 100-150 kb in length) of genomic DNA from the species of interest. The library is screened by probe hybridization or PCR to identify clones that contain the flanking marker sequences. Then a series of partially overlapping clones that connects the two flanking clones (a “contig”) is built up through physical mapping procedures. These procedures include fingerprinting, STS content mapping and sequence-tagged connector methodologies. Once the physical contig is constructed and sequenced, the sequence is searched for all transcriptional units. The transcriptional unit that corresponds to the trait gene can be determined by comparing sequences between mutant and wild type strains, by additional fine-scale genetic mapping, and/or by functional testing through plant transformation. Trait genes identified in this way become leads for transgenic product development. Similarly, trait genes identified by association studies with candidate genes become leads for transgenic product development.

3. Marker-Aided Breeding and Marker-Assisted Selection

When a trait gene has been localized in the vicinity of genetic markers, those markers can be used to select for improved values of the trait without the need for phenotypic analysis at each cycle of selection. In marker-aided breeding and marker-assisted selection, associations between trait genes and markers are established initially through genetic mapping analysis (as in M.1 or M.2). In the same process, one determines which marker alleles are linked to favorable trait gene alleles. Subsequently, marker alleles associated with favorable trait gene alleles are selected in the population. This procedure will improve the value of the trait provided that there is sufficiently close linkage between markers and trait genes. The degree of linkage required depends upon the number of generations of selection because, at each generation, there is opportunity for breakdown of the association through recombination.

4. Prediction of Crosses for New Inbred Line Development

The associations between specific marker alleles and favorable trait gene alleles also can be used to predict what types of progeny may segregate from a given cross. This prediction may allow selection of appropriate parents to generation populations from which new combinations of favorable trait gene alleles are assembled to produce a new inbred line. For example, if line A has marker alleles previously known to be associated with favorable trait alleles at loci 1, 20 and 31, while line B has marker alleles associated with favorable effects at loci 15, 27 and 29, then a new line could be developed by crossing A x B and selecting progeny that have favorable alleles at all 6 trait loci.

5. Hybrid Prediction

Commercial corn seed is produced by making hybrids between two elite inbred lines that belong to different “heterotic groups”. These groups are sufficiently distinct genetically that hybrids between them show high levels of heterosis or hybrid vigor (i.e., increased performance relative to the parental lines). By analyzing the marker constitution of good hybrids, one can identify sets of alleles at different loci in both male and female lines that combine well to produce heterosis. Understanding these patterns, and knowing the marker constitution of different inbred lines, can allow prediction of the level of heterosis between different pairs of lines. These predictions can narrow down the possibilities of which line(s) of opposite heterotic group should be used to test the performance of a new inbred line.

6. Identity by Descent

One theory of heterosis predicts that regions of identity by descent (IBD) between the male and female lines used to produce a hybrid will reduce hybrid performance. Identity by descent can be inferred from patterns of marker alleles in different lines. An identical string of markers at a series of adjacent loci may be considered identical by descent if it is unlikely to occur independently by chance. Analysis of marker fingerprints in male and female lines can identify regions of IBD. Knowledge of these regions can inform the choice of hybrid parents, because avoiding IBD in hybrids is likely to improve performance. This knowledge may also inform breeding programs in that crosses could be designed to produce pairs of inbred lines (one male and one female) that show little or no IBD.

A fingerprint of an inbred line is the combination of alleles at a set of marker loci. High density fingerprints can be used to establish and trace the identity of germplasm, which has utility in germplasm ownership protection.

Genetic markers are used to accelerate introgression of transgenes into new genetic backgrounds (i.e., into a diverse range of germplasm). Simple introgression involves crossing a transgenic line to an elite inbred line and then backcrossing the hybrid repeatedly to the elite (recurrent) parent, while selecting for maintenance of the transgene. Over multiple backcross generations, the genetic background of the original transgenic line is replaced gradually by the genetic background of the elite inbred through recombination and segregation. This process can be accelerated by selection on marker alleles that derive from the recurrent parent.

Use of Polymorphism Assay for Mapping a Library of DNA Clones

The polymorphisms and loci of this invention are useful for identifying and mapping DNA sequence of QTLs and genes linked to the polymorphisms. For instance, BAC or YAC clone libraries can be queried using polymorphisms linked to a trait to find a clone containing specific QTLs and genes associated with the trait. For instance, QTLs and genes in a plurality, e.g., hundreds or thousands, of large, multi-gene sequences can be identified by hybridization with an oligonucleotide probe that hybridizes to a mapped and/or linked polymorphism. Such hybridization screening can be improved by providing clone sequence in a high density array. The screening method is more preferably enhanced by employing a pooling strategy to significantly reduce the number of hybridizations required to identify a clone containing the polymorphism. When the polymorphisms are mapped, the screening effectively maps the clones.

For instance, in a case where thousands of clones are arranged in a defined array, e.g., in 96-well plates, the plates can be arbitrarily arranged in three-dimensionally, arrayed stacks of wells each comprising a unique DNA clone. The wells in each stack can be represented as discrete elements in a three dimensional array of rows, columns and plates. In one aspect of the invention the number of stacks and plates in a stack are about equal to minimize the number of assays. The stacks of plates allow the construction of pools of cloned DNA.

For a three-dimensionally arrayed stack, pools of cloned DNA can be created for (a) all of the elements in each row, (b) all of the elements of each column, and (c) all of the elements of each plate. Hybridization screening of the pools with an oligonucleotide probe that hybridizes to a polymorphism unique to one of the clones will provide a positive indication for one column pool, one row pool and one plate pool, thereby indicating the well element containing the target clone.

In the case of multiple stacks, additional pools of all of the clone DNA in each stack allows indication of the stack having the row-column-plate coordinates of the target clone. For instance, a 4608 clone set can be disposed in 48 96-well plates. The 48 plates can be arranged in 8 sets of 6-plate stacks providing 6x12x8 three-dimensional arrays of elements, i.e., each stack comprises 6 stacks of 8 rows and 12 columns. For the entire clone set there are 36 pools, i.e., 6 stack pools, 8 row pools, 12 column pools and 8 stack pools. Thus, a maximum of 36 hybridization reactions is required to find the clone harboring QTLs or genes associated or linked to each mapped polymorphism.

Once a clone is identified, genes within that clone can be tested for whether they affect the trait by analysis of recombinants in a mapping population, further linkage disequilibrium analysis, and ultimately transgenic testing. Additional genes can be identified by finding additional clones overlapping the one containing the original polymorphism through contig building, as described above.

Breeding Plants of the Invention

In addition to direct transformation of a particular plant genotype with a construct prepared according to the current invention, transgenic plants may be made by crossing a plant having a construct of the invention to a second plant lacking the construct. For example, a selected coding region operably linked to a promoter can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the current invention not only encompasses a plant directly regenerated from cells that have been transformed in accordance with the current invention, but also the progeny of such plants. As used herein the term “progeny” denotes the offspring of any generation of a parent plant prepared in accordance with the instant invention, wherein the progeny comprises a construct prepared in accordance with the invention. “Crossing” a plant to provide a plant line having one or more added transgenes relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a transgene of the invention being introduced into a plant line by crossing a starting line with a donor plant line that comprises a transgene of the invention. To achieve this one could, for example, perform the following steps:

-   (a) plant seeds of the first (starting line) and second (donor plant     line that comprises a transgene of the invention) parent plants; -   (b) grow the seeds of the first and second parent plants into plants     that bear flowers; -   (c) pollinate a flower from the first parent plant with pollen from     the second parent plant; and -   (d) harvest seeds produced on the parent plant bearing the     fertilized flower.     Backcrossing is herein defined as the process including the steps     of: -   (a) crossing a plant of a first genotype containing a desired gene,     DNA sequence or element to a plant of a second genotype lacking the     desired gene, DNA sequence or element; -   (b) selecting one or more progeny plants containing the desired     gene, DNA sequence or element; -   (c) crossing the progeny plant to a plant of the second genotype;     and -   (d) repeating steps (b) and (c) for the purpose of transferring the     desired gene, DNA sequence or element from a plant of a first     genotype to a plant of a second genotype.

Plant Breeding

Introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion. A plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly a plant genotype lacking the desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.

Backcrossing can be used to improve a starting plant. Backcrossing transfers a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent, then the selected progeny are mated back to the superior recurrent parent (A). After five or more backcross generations with selection for the desired trait, the progeny are hemizygous for loci controlling the characteristic being transferred but are like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give progeny that are pure breeding for the gene(s) being transferred, i.e., one or more transformation events.

Therefore, through a series a breeding manipulations, a selected transgene may be moved from one line into an entirely different line without the need for further recombinant manipulation. Transgenes are valuable in that they typically behave genetically as any other gene and can be manipulated by breeding techniques in a manner identical to any other corn gene. Therefore, one may produce inbred plants that are true breeding for one or more transgenes. By crossing different inbred plants, one may produce a large number of different hybrids with different combinations of transgenes. In this way, plants may be produced that have the desirable agronomic properties frequently associated with hybrids (“hybrid vigor”), as well as the desirable characteristics imparted by one or more transgene(s).

It is desirable to introgress the genes of the present invention into maize hybrids for characterization of the phenotype conferred by each gene in a transformed plant. The host genotype into which the transgene was introduced, preferably LH59, is an elite inbred and therefore only limited breeding is necessary in order to produce high yielding maize hybrids. The transformed plant, regenerated from callus is crossed, to the same genotype, e.g., LH59. The progeny are self-pollinated twice, and plants homozygous for the transgene are identified. Homozygous transgenic plants are crossed to a testcross parent in order to produce hybrids. The test cross parent is an inbred belonging to a heterotic group that is different from that of the transgenic parent and for which it is known that high yielding hybrids can be generated, for example hybrids are produced from crosses of LH159 to either LH195 or LH200.

The following examples illustrate the identification of polymorphic markers useful for mapping and isolating genes of this invention and as markers of QTLs and genes associated with an oil-related trait. Other examples illustrate the identification of oil-related genes and partial genes. Still other examples illustrate methods for inserting genes of this invention into a plant expression vector, i.e., operably linked to a promoter and other regulatory elements, to confer an oil-related trait to a transgenic plant.

EXAMPLE 1

This example illustrates the identification of polymorphic maize markers of this invention.

A set of more than 800 candidate oil genes was identified (a) as homologs of plant genes that are believed to be in an oil-related metabolic pathway of a model plant such as Arabidopsis thaliana; (b) by comparing transcription profiling results for high oil and low oil maize lines; and (c) by subtractive hybridization between endosperm tissues of high oil and low oil lines. The sequences of the candidate oil genes were queried against a proprietary collection of maize genes and partial maize genes, e.g., genomic sequence or ESTs, to identify a set of DNA sequences for candidate maize markers.

Maize polymorphisms were identified by comparing alignments of DNA sequences from separate maize lines. Candidate polymorphisms were qualified by the following parameters:

-   The minimum length of sequence for a synthetic reference sequence is     200 bases. -   The percentage identity of observed bases in a region of 15 bases on     each side of a candidate SNP, is 75%. -   The minimum BLAST quality in each of the various sequences at a     polymorphism site is 35. -   The minimum BLAST quality in a region of 15 bases on each side of     the polymorphism site is 20.

The SNP and Indel polymorphisms in each locus were qualified for detection by development of an assay, e.g., Taqman® assay (Applied Biosystems, Foster City, Calif.). Assay qualified polymorphisms are evaluated for oil informativeness by comparing allelic frequencies in the two parental lines of an association study population. The parent lines were an oil rich maize line and an oil poor maize line, i.e., the University of Illinois High Oil and Low Oil maize lines as described by Dudley and Lambert (1992, Maydica 37: 81-87).

Informativeness is reported as an allelic frequency difference between parental populations, i.e. the high oil line and the low oil line. When one of the parents, e.g., the high oil line, is fixed, its allelic frequency is 1. Markers were qualified if they had an allelic frequency difference of at least 0.6. If the marker was fixed on either parent with a frequency of 0 or 1, a marker could be selected at a lower allelic frequency difference of at least 0.4. The informative markers were viewed on a genetic map to identify marker-deficient regions of chromosomes. Markers with lower allelic frequency difference, e.g., as low as 0.15, were selected to fill in the marker-deficient regions of chromosomes.

DNA amplicons of 484 polymorphic maize genomic DNA loci of this invention are provided in the sequence listing as SEQ ID NO:1 through SEQ ID NO:484. Table 1 provides a description of polymorphisms in the 484 DNA amplicons. Particular aspects of the markers are identified in Table 1 by reference to:

SEQ_NUM, which refers to the sequence number of a nucleic acid sequence in the sequence listing, e.g. SEQ ID NO:1; and

SEQ_ID, which refers to an arbitrary identifying name for an amplicon of a polymorphic locus, e.g. “Amplicon25”;

MUTATION_ID, which refers to one or more arbitrary identifying names for each polymorphism, e.g. “91”;

START_POS which refers to the position in the nucleotide sequence of the polymorphic maize DNA locus where the polymorphism begins, e.g. 110;

END_POS which refers to the position in the nucleotide sequence of the polymorphic maize DNA locus where the polymorphism ends; for SNPs the START_POS and END_POS are common;

TYPE which refers to the identification of the polymorphism as an SNP or IND (Indel);

ALLELEn and STRAINn which refer to the nucleotide sequence of a polymorphism in a specific allelic maize variety, e.g. “C”, “T”, and when strains are indicated “mol7” and “b73”;

Taqman® assays for 488 markers in the 484 polymorphic loci (4 loci are represented by marker assays for 2 separate polymorphisms) are characterized by four separate DNA molecules, i.e. a forward PCR primer, a reverse PCR primer, a VIC-labeled hybridization probe and a FAM-labeled hybridization probe, identified by SEQ ID NO:485 through SEQ ID NO:2436. The primers and probes for each of the 488 markers are more particularly identified in Table 2 by reference to:

Chromosome which refers to one of ten maize chromosomes.

Position which refers to distance to the marker measured in cM from the 5′ end of the chromosome.

Marker which corresponds to the MUTATION_ID in the amplicon of SEQ_NUM (1-484).

Forward designates the number in the Sequence Listing for the DNA sequence of the forward primer.

Reverse designates the number in the Sequence Listing for the DNA sequence of the reverse primer.

FAM designates the number in the Sequence Listing for the FAM-labeled hybridization probe.

VIC designates the number in the Sequence Listing for the VIC-labeled hybridization probe.

TABLE 2 Chro- mo- some Position Marker SEQ_NUM Forward Reverse FAM VIC 1 3.7 111829 406 2121 2122 2123 2124 1 5.6 147181 442 2265 2266 2267 2268 1 14.9 36199 222 1381 1382 1383 1384 1 17.3 25418 149 1081 1082 1083 1084 1 22.4 28164 155 1105 1106 1107 1108 1 25.1 43230 269 1573 1574 1575 1576 1 30.4 2847 11 525 526 527 528 1 30.4 144506 424 2193 2194 2195 2196 1 40.2 36685 228 1405 1406 1407 1408 1 44 104827 336 1841 1842 1843 1844 1 45 151360 459 2333 2334 2335 2336 1 46.8 35417 216 1353 1354 1355 1356 1 46.8 37716 236 1437 1438 1439 1440 1 47.1 4409 25 585 586 587 588 1 52.2 42173 266 1561 1562 1563 1564 1 58.4 116 2 489 490 491 492 1 58.4 9159 52 693 694 695 696 1 60.3 143100 412 2145 2146 2147 2148 1 60.5 16876 113 937 938 939 940 1 60.6 33819 206 1313 1314 1315 1316 1 60.6 40124 254 1513 1514 1515 1516 1 60.6 40189 255 1517 1518 1519 1520 1 62.5 9449 57 713 714 715 716 1 62.5 33372 203 1301 1302 1303 1304 1 68.8 148156 451 2301 2302 2303 2304 1 83.2 25863 147 1073 1074 1075 1076 1 83.2 34205 207 1317 1318 1319 1320 1 83.2 43789 271 1581 1582 1583 1584 1 83.7 11522 74 781 782 783 784 1 83.7 106244 348 1889 1890 1891 1892 1 85.6 144090 423 2189 2190 2191 2192 1 85.9 5215 35 625 626 627 628 1 85.9 27375 152 1093 1094 1095 1096 1 85.9 69188 301 1701 1702 1703 1704 1 86.3 8984 49 681 682 683 684 1 86.3 36286 223 1385 1386 1387 1388 1 86.6 148194 452 2305 2306 2307 2308 1 88.8 29829 170 1165 1166 1167 1168 1 88.8 37068 231 1417 1418 1419 1420 1 88.8 68435 297 1685 1686 1687 1688 1 89.6 111365 399 2093 2094 2095 2096 1 90.5 60430 293 1669 1670 1671 1672 1 90.5 111828 405 2117 2118 2119 2120 1 91 113263 410 2137 2138 2139 2140 1 91.8 104474 333 1829 1830 1831 1832 1 92.3 145573 433 2229 2230 2231 2232 1 95 39351 247 1485 1486 1487 1488 1 96.4 107701 364 1953 1954 1955 1956 1 96.9 36448 225 1393 1394 1395 1396 1 99 40655 261 1541 1542 1543 1544 1 99 107077 357 1925 1926 1927 1928 1 103.3 8719 40 645 646 647 648 1 116.3 40338 257 1525 1526 1527 1528 1 116.3 54410 284 1633 1634 1635 1636 1 121.1 107621 362 1945 1946 1947 1948 1 121.5 16755 111 929 930 931 932 1 121.5 36863 230 1413 1414 1415 1416 1 122.1 41280 263 1549 1550 1551 1552 1 123.3 109328 379 2013 2014 2015 2016 1 124.6 33373 204 1305 1306 1307 1308 1 127.6 105648 346 1881 1882 1883 1884 1 129.5 4453 28 597 598 599 600 1 129.5 9626 64 741 742 743 744 1 129.5 37689 235 1433 1434 1435 1436 1 130.3 69565 302 1705 1706 1707 1708 1 132.1 34903 209 1325 1326 1327 1328 1 133.9 16724 102 893 894 895 896 1 138.5 12824 79 801 802 803 804 1 139.2 38701 241 1461 1462 1463 1464 1 140.8 5098 33 617 618 619 620 1 153.7 31993 191 1253 1254 1255 1256 1 156.7 8982 48 677 678 679 680 1 159.7 39502 248 1489 1490 1491 1492 1 160.4 148362 455 2317 2318 2319 2320 1 164.2 39896 252 1505 1506 1507 1508 1 165.6 108862 376 2001 2002 2003 2004 1 168.3 9701 67 753 754 755 756 1 178.6 151382 457 2325 2326 2327 2328 1 179.8 32253 198 1281 1282 1283 1284 1 194.2 13490 81 809 810 811 812 1 200.3 30840 181 1213 1214 1215 1216 1 207 16137 98 877 878 879 880 2 5.8 9867 71 769 770 771 772 2 5.8 31064 183 1221 1222 1223 1224 2 12.9 104447 332 1825 1826 1827 1828 2 14.1 39289 246 1481 1482 1483 1484 2 17.5 106678 351 1901 1902 1903 1904 2 19.5 82235 325 1797 1798 1799 1800 2 30.1 106842 353 1909 1910 1911 1912 2 32.8 2945 12 529 530 531 532 2 32.8 16074 97 873 874 875 876 2 33.9 80031 321 1781 1782 1783 1784 2 35.9 13691 89 841 842 843 844 2 35.9 50315 277 1605 1606 1607 1608 2 38.3 9706 68 757 758 759 760 2 42.4 32016 192 1257 1258 1259 1260 2 62.2 80704 323 1789 1790 1791 1792 2 70.4 9364 56 709 710 711 712 2 74.8 9623 63 737 738 739 740 2 75.6 40931 262 1545 1546 1547 1548 2 76.2 36323 224 1389 1390 1391 1392 2 76.2 104946 340 1857 1858 1859 1860 2 77.4 111617 403 2109 2110 2111 2112 2 78.2 11466 73 777 778 779 780 2 78.2 79073 315 1757 1758 1759 1760 2 78.2 108493 374 1993 1994 1995 1996 2 87.3 23442 143 1057 1058 1059 1060 2 87.5 107911 368 1969 1970 1971 1972 2 92.5 551 4 497 498 499 500 2 92.5 3177 14 537 538 539 540 2 92.5 53097 282 1625 1626 1627 1628 2 92.9 366 3 493 494 495 496 2 92.9 84829 330 1817 1818 1819 1820 2 99.7 151288 458 2329 2330 2331 2332 2 104.8 82458 327 1805 1806 1807 1808 2 106 111475 402 2105 2106 2107 2108 2 106.2 108013 371 1981 1982 1983 1984 2 107.6 2307 8 513 514 515 516 2 114.9 22775 135 1025 1026 1027 1028 2 123.4 104954 341 1861 1862 1863 1864 2 127 41850 264 1553 1554 1555 1556 2 134.9 31474 187 1237 1238 1239 1240 2 139.8 109207 377 2005 2006 2007 2008 2 144.2 35297 212 1337 1338 1339 1340 2 152.4 43579 270 1577 1578 1579 1580 2 153.5 147548 448 2289 2290 2291 2292 2 156.6 14467 84 821 822 823 824 2 157.2 33320 202 1297 1298 1299 1300 2 164.2 735 7 509 510 511 512 2 164.2 76792 308 1729 1730 1731 1732 3 6 8911 45 665 666 667 668 3 6 51614 280 1617 1618 1619 1620 3 9.1 10667 72 773 774 775 776 3 19.7 19963 117 953 954 955 956 3 19.7 32137 196 1273 1274 1275 1276 3 46.2 49293 275 1597 1598 1599 1600 3 52.3 109315 378 2009 2010 2011 2012 3 53.5 25000 144 1061 1062 1063 1064 3 54.1 21154 125 985 986 987 988 3 54.1 109722 384 2033 2034 2035 2036 3 57.2 109509 382 2025 2026 2027 2028 3 57.2 146158 435 2237 2238 2239 2240 3 57.5 107784 365 1957 1958 1959 1960 3 58.6 29867 173 1177 1178 1179 1180 3 59.3 4599 30 605 606 607 608 3 59.3 21190 131 1009 1010 1011 1012 3 59.3 28923 159 1121 1122 1123 1124 3 59.3 83776 328 1809 1810 1811 1812 3 59.3 147511 447 2285 2286 2287 2288 3 59.3 147768 449 2293 2294 2295 2296 3 60.4 8685 39 641 642 643 644 3 60.4 9468 58 717 718 719 720 3 60.4 9470 59 721 722 723 724 3 60.5 145322 432 2225 2226 2227 2228 3 61 16729 103 897 898 899 900 3 61.7 32247 197 1277 1278 1279 1280 3 61.7 39785 251 1501 1502 1503 1504 3 62.7 9144 51 689 690 691 692 3 62.7 9739 69 761 762 763 764 3 68.5 153431 460 2337 2338 2339 2340 3 68.5 154505 474 2393 2394 2395 2396 3 68.5 154509 477 2405 2406 2407 2408 3 68.5 154511 475 2397 2398 2399 2400 3 68.5 154532 472 2385 2386 2387 2388 3 68.5 154536 469 2373 2374 2375 2376 3 68.5 154552 471 2381 2382 2383 2384 3 68.5 154616 473 2389 2390 2391 2392 3 68.5 155689 479 2413 2414 2415 2416 3 68.5 155708 480 2417 2418 2419 2420 3 71 4886 31 609 610 611 612 3 71.5 24395 141 1049 1050 1051 1052 3 71.8 79081 316 1761 1762 1763 1764 3 74.3 23890 145 1065 1066 1067 1068 3 79.2 9173 53 697 698 699 700 3 89.7 15954 93 857 858 859 860 3 89.7 15965 94 861 862 863 864 3 93.2 77118 309 1733 1734 1735 1736 3 96.1 21772 129 1001 1002 1003 1004 3 96.5 36694 229 1409 1410 1411 1412 3 98.2 111204 397 2085 2086 2087 2088 3 98.6 29390 165 1145 1146 1147 1148 3 101.3 108630 375 1997 1998 1999 2000 3 105 35568 217 1357 1358 1359 1360 3 106.9 9473 60 725 726 727 728 3 109.4 21603 127 993 994 995 996 3 111.4 110780 392 2065 2066 2067 2068 3 118.4 146534 439 2253 2254 2255 2256 3 118.9 56939 287 1645 1646 1647 1648 3 123.8 143969 422 2185 2186 2187 2188 3 127.7 9079 50 685 686 687 688 3 139.1 3970 22 573 574 575 576 4 1 12340 77 793 794 795 796 4 23.6 2739 10 521 522 523 524 4 38.7 110069 386 2041 2042 2043 2044 4 38.7 111464 400 2097 2098 2099 2100 4 52.8 24647 146 1069 1070 1071 1072 4 53.2 40461 259 1533 1534 1535 1536 4 53.2 156243 484 2433 2434 2435 2436 4 58.6 1122 6 505 506 507 508 4 58.6 12012 75 785 786 787 788 4 62.1 10671 76 789 790 791 792 4 63.2 70730 305 1717 1718 1719 1720 4 64.9 38852 242 1465 1466 1467 1468 4 67.6 15096 90 845 846 847 848 4 67.6 154038 467 2365 2366 2367 2368 4 69.5 3351 19 561 562 563 564 4 69.5 5021 32 613 614 615 616 4 69.5 14666 88 837 838 839 840 4 69.5 15247 100 885 886 887 888 4 69.5 37503 232 1421 1422 1423 1424 4 69.5 80475 322 1785 1786 1787 1788 4 69.5 153424 464 2353 2354 2355 2356 4 69.9 107276 358 1929 1930 1931 1932 4 71.4 84527 329 1813 1814 1815 1816 4 73 35294 211 1333 1334 1335 1336 4 80 106845 354 1913 1914 1915 1916 4 86.5 3964 21 569 570 571 572 4 87.7 107840 366 1961 1962 1963 1964 4 92.1 9187 54 701 702 703 704 4 107.7 106491 350 1897 1898 1899 1900 4 108.2 39511 249 1493 1494 1495 1496 4 109.2 23289 138 1037 1038 1039 1040 4 109.7 26846 150 1085 1086 1087 1088 4 109.7 28933 161 1129 1130 1131 1132 4 110.3 8979 47 673 674 675 676 4 112.4 54460 285 1637 1638 1639 1640 4 115.1 71159 306 1721 1722 1723 1724 4 115.4 29435 166 1149 1150 1151 1152 4 117.6 30745 179 1205 1206 1207 1208 4 119.2 2435 9 517 518 519 520 4 119.2 12711 78 797 798 799 800 4 119.2 17828 108 917 918 919 920 4 119.2 18439 115 945 946 947 948 4 119.2 20933 121 969 970 971 972 4 119.2 20934 122 973 974 975 976 4 119.2 24422 142 1053 1054 1055 1056 4 120.9 29194 163 1137 1138 1139 1140 4 122.4 151472 456 2321 2322 2323 2324 4 128.1 32049 195 1269 1270 1271 1272 4 133.6 3224 16 545 546 547 548 4 133.6 3226 16 549 550 551 552 4 135.1 3152 13 533 534 535 536 4 135.1 4445 27 593 594 595 596 4 135.1 13833 82 813 814 815 816 4 135.8 17900 109 921 922 923 924 4 136.4 147219 443 2269 2270 2271 2272 4 142.1 147037 441 2261 2262 2263 2264 4 143.1 43121 268 1569 1570 1571 1572 4 144.8 35338 213 1341 1342 1343 1344 5 1.6 24265 140 1045 1046 1047 1048 5 1.6 31790 190 1249 1250 1251 1252 5 9.7 143251 414 2153 2154 2155 2156 5 13.8 69592 303 1709 1710 1711 1712 5 16.7 57137 288 1649 1650 1651 1652 5 16.7 105613 345 1877 1878 1879 1880 5 17.2 107858 367 1965 1966 1967 1968 5 27.4 91 1 485 486 487 488 5 31.3 5275 36 629 630 631 632 5 39.9 109403 381 2021 2022 2023 2024 5 41.7 16527 99 881 882 883 884 5 50.9 109342 380 2017 2018 2019 2020 5 51.9 16762 104 901 902 903 904 5 51.9 16767 105 905 906 907 908 5 53.4 79519 318 1769 1770 1771 1772 5 56.5 9668 66 749 750 751 752 5 57.7 30270 177 1197 1198 1199 1200 5 57.7 52081 281 1621 1622 1623 1624 5 57.7 77545 311 1741 1742 1743 1744 5 62.3 51419 279 1613 1614 1615 1616 5 63 32272 199 1285 1286 1287 1288 5 66.9 30000 174 1181 1182 1183 1184 5 66.9 146415 437 2245 2246 2247 2248 5 69.3 106912 355 1917 1918 1919 1920 5 69.6 107061 356 1921 1922 1923 1924 5 69.6 144731 426 2201 2202 2203 2204 5 70.5 105854 347 1885 1886 1887 1888 5 70.8 22796 136 1029 1030 1031 1032 5 70.8 27874 158 1117 1118 1119 1120 5 71.7 143216 413 2149 2150 2151 2152 5 73.2 33249 201 1293 1294 1295 1296 5 76.4 29820 169 1161 1162 1163 1164 5 78.2 144687 425 2197 2198 2199 2200 5 80.2 36637 227 1401 1402 1403 1404 5 80.9 143418 419 2173 2174 2175 2176 5 81.2 38478 238 1449 1450 1451 1452 5 81.5 48616 274 1593 1594 1595 1596 5 83 104850 337 1845 1846 1847 1848 5 83.7 148026 450 2297 2298 2299 2300 5 86.2 9297 55 705 706 707 708 5 87.3 106300 349 1893 1894 1895 1896 5 90.6 5480 38 637 638 639 640 5 100.9 35377 214 1345 1346 1347 1348 5 104.5 58375 290 1657 1658 1659 1660 5 117.1 143380 416 2161 2162 2163 2164 5 136 105546 343 1869 1870 1871 1872 5 150.5 31084 184 1225 1226 1227 1228 6 16.4 27615 157 1113 1114 1115 1116 6 17.3 154854 476 2401 2402 2403 2404 6 19.4 105014 342 1865 1866 1867 1868 6 22.9 79529 319 1773 1774 1775 1776 6 30.5 3284 18 557 558 559 560 6 30.5 69630 304 1713 1714 1715 1716 6 32.8 29780 168 1157 1158 1159 1160 6 32.8 68941 298 1689 1690 1691 1692 6 32.8 146458 438 2249 2250 2251 2252 6 33.3 104510 334 1833 1834 1835 1836 6 33.3 107639 363 1949 1950 1951 1952 6 35.3 15304 101 889 890 891 892 6 35.3 16944 106 909 910 911 912 6 35.3 35574 218 1361 1362 1363 1364 6 35.3 77413 310 1737 1738 1739 1740 6 35.3 110607 389 2053 2054 2055 2056 6 37.3 36067 221 1373 1374 1375 1376 6 37.3 36073 221 1377 1378 1379 1380 6 41.2 34560 208 1321 1322 1323 1324 6 43.1 30176 176 1193 1194 1195 1196 6 52.8 4463 29 601 602 603 604 6 53.1 60751 294 1673 1674 1675 1676 6 53.5 32034 194 1265 1266 1267 1268 6 53.5 57758 289 1653 1654 1655 1656 6 53.5 108212 372 1985 1986 1987 1988 6 58.1 59008 292 1665 1666 1667 1668 6 58.1 146195 436 2241 2242 2243 2244 6 59.9 3277 17 553 554 555 556 6 59.9 105586 344 1873 1874 1875 1876 6 61.5 148039 453 2309 2310 2311 2312 6 61.5 155861 482 2425 2426 2427 2428 6 63.1 20410 128 997 998 999 1000 6 66.6 8838 43 657 658 659 660 6 67.5 14694 96 869 870 871 872 6 73.3 113381 411 2141 2142 2143 2144 6 86.9 110972 395 2077 2078 2079 2080 6 92.8 37947 237 1441 1442 1443 1444 6 92.8 37948 237 1445 1446 1447 1448 6 93.9 5319 37 633 634 635 636 6 95.8 30771 180 1209 1210 1211 1212 6 97.8 27295 151 1089 1090 1091 1092 6 99.1 108433 373 1989 1990 1991 1992 6 110.4 31684 189 1245 1246 1247 1248 6 112.4 107449 361 1941 1942 1943 1944 6 117.4 16017 107 913 914 915 916 6 121 37634 234 1429 1430 1431 1432 6 128.7 9667 65 745 746 747 748 6 130.8 21433 126 989 990 991 992 6 132.7 37555 233 1425 1426 1427 1428 7 56.2 68954 299 1693 1694 1695 1696 7 62 31370 186 1233 1234 1235 1236 7 62 42164 265 1557 1558 1559 1560 7 67 30674 178 1201 1202 1203 1204 7 67.4 33769 205 1309 1310 1311 1312 7 67.9 39064 244 1473 1474 1475 1476 7 69.1 35633 219 1365 1366 1367 1368 7 72.8 42930 267 1565 1566 1567 1568 7 73.8 68426 296 1681 1682 1683 1684 7 73.9 29005 162 1133 1134 1135 1136 7 74 51405 278 1609 1610 1611 1612 7 83.6 29362 164 1141 1142 1143 1144 7 98.5 8799 42 653 654 655 656 7 98.8 48425 272 1585 1586 1587 1588 7 99.8 4415 26 589 590 591 592 7 99.8 35408 215 1349 1350 1351 1352 7 103.6 79695 320 1777 1778 1779 1780 7 107.5 38914 243 1469 1470 1471 1472 7 109.5 39978 253 1509 1510 1511 1512 7 113.3 155829 481 2421 2422 2423 2424 7 114.7 28932 160 1125 1126 1127 1128 7 114.7 31547 188 1241 1242 1243 1244 7 114.7 68149 295 1677 1678 1679 1680 7 115.8 4093 23 577 578 579 580 7 118.6 4302 24 581 582 583 584 7 118.6 38653 240 1457 1458 1459 1460 7 118.6 78828 314 1753 1754 1755 1756 7 118.6 81460 324 1793 1794 1795 1796 7 120.9 153856 463 2349 2350 2351 2352 7 122.2 145260 429 2213 2214 2215 2216 7 124.5 15184 92 853 854 855 856 7 124.5 39773 250 1497 1498 1499 1500 7 131.6 79307 317 1765 1766 1767 1768 7 132.8 30026 175 1185 1186 1187 1188 7 132.8 30029 175 1189 1190 1191 1192 7 144.6 30872 182 1217 1218 1219 1220 7 154.5 110771 391 2061 2062 2063 2064 7 164.7 155475 478 2409 2410 2411 2412 7 165.5 146593 440 2257 2258 2259 2260 7 167 143371 415 2157 2158 2159 2160 7 186.5 36490 226 1397 1398 1399 1400 7 189.3 18157 112 933 934 935 936 7 189.4 21038 124 981 982 983 984 7 189.4 69120 300 1697 1698 1699 1700 7 189.4 71624 307 1725 1726 1727 1728 7 193.9 19704 123 977 978 979 980 8 8.8 35173 210 1329 1330 1331 1332 8 16.4 40320 256 1521 1522 1523 1524 8 16.9 19198 116 949 950 951 952 8 34.3 29842 172 1173 1174 1175 1176 8 40.9 107937 369 1973 1974 1975 1976 8 43.1 111628 404 2113 2114 2115 2116 8 43.3 26720 154 1101 1102 1103 1104 8 45.5 32030 193 1261 1262 1263 1264 8 47.3 53899 283 1629 1630 1631 1632 8 47.9 104862 339 1853 1854 1855 1856 8 49.7 107396 360 1937 1938 1939 1940 8 53.9 27361 156 1109 1110 1111 1112 8 53.9 145200 428 2209 2210 2211 2212 8 55.7 23091 137 1033 1034 1035 1036 8 59.3 77568 312 1745 1746 1747 1748 8 64 110148 387 2045 2046 2047 2048 8 64.5 104858 338 1849 1850 1851 1852 8 65.8 104389 331 1821 1822 1823 1824 8 66.6 21895 130 1005 1006 1007 1008 8 67.4 48562 273 1589 1590 1591 1592 8 68.4 82295 326 1801 1802 1803 1804 8 76.8 111472 401 2101 2102 2103 2104 8 85.9 110684 390 2057 2058 2059 2060 8 87.5 9759 70 765 766 767 768 8 87.5 20537 120 965 966 967 968 8 87.5 112497 408 2129 2130 2131 2132 8 105.5 107286 359 1933 1934 1935 1936 8 106.8 13100 80 805 806 807 808 8 117.3 145077 427 2205 2206 2207 2208 8 117.3 145298 430 2217 2218 2219 2220 8 132.8 14545 87 833 834 835 836 8 135.5 8757 41 649 650 651 652 8 137.4 561 5 501 502 503 504 9 0 14479 86 829 830 831 832 9 20.5 58904 291 1661 1662 1663 1664 9 38.1 49557 276 1601 1602 1603 1604 9 78.2 29745 167 1153 1154 1155 1156 9 87.7 12557 83 817 818 819 820 9 90.3 23779 139 1041 1042 1043 1044 9 93.4 29832 171 1169 1170 1171 1172 9 93.4 55370 286 1641 1642 1643 1644 9 93.8 110377 388 2049 2050 2051 2052 9 93.8 113113 409 2133 2134 2135 2136 9 93.9 155159 470 2377 2378 2379 2380 9 94 25961 148 1077 1078 1079 1080 9 94.5 148621 454 2313 2314 2315 2316 9 94.6 112139 407 2125 2126 2127 2128 9 94.7 31233 185 1229 1230 1231 1232 9 94.7 153885 465 2357 2358 2359 2360 9 100.6 20048 118 957 958 959 960 9 100.6 153427 461 2341 2342 2343 2344 9 108.3 109802 385 2037 2038 2039 2040 9 108.9 155793 483 2429 2430 2431 2432 9 110.3 8937 46 669 670 671 672 9 110.3 78438 313 1749 1750 1751 1752 9 111.2 147496 446 2281 2282 2283 2284 9 112.4 13086 85 825 826 827 828 9 115 145318 431 2221 2222 2223 2224 9 125.2 9555 62 733 734 735 736 9 137.2 36022 220 1369 1370 1371 1372 9 145.8 110800 393 2069 2070 2071 2072 9 165.8 110886 394 2073 2074 2075 2076 9 171.4 147417 445 2277 2278 2279 2280 10 23.9 153632 462 2345 2346 2347 2348 10 23.9 153987 466 2361 2362 2363 2364 10 23.9 154021 468 2369 2370 2371 2372 10 29.7 20502 119 961 962 963 964 10 31.7 104672 335 1837 1838 1839 1840 10 36.9 111004 396 2081 2082 2083 2084 10 43.4 16041 95 865 866 867 868 10 50.5 143408 418 2169 2170 2171 2172 10 51.1 147411 444 2273 2274 2275 2276 10 52.7 143754 421 2181 2182 2183 2184 10 54.7 5140 34 621 622 623 624 10 55.6 111212 398 2089 2090 2091 2092 10 55.8 8840 44 661 662 663 664 10 55.8 21292 132 1013 1014 1015 1016 10 55.8 22541 133 1017 1018 1019 1020 10 55.8 143388 417 2165 2166 2167 2168 10 56.1 39275 245 1477 1478 1479 1480 10 56.7 22717 134 1021 1022 1023 1024 10 57 3206 15 541 542 543 544 10 57 32428 200 1289 1290 1291 1292 10 65.5 3640 20 565 566 567 568 10 66.5 16730 110 925 926 927 928 10 68 107941 370 1977 1978 1979 1980 10 71.6 18392 114 941 942 943 944 10 73.6 27447 153 1097 1098 1099 1100 10 77.4 13745 91 849 850 851 852 10 83.9 38604 239 1453 1454 1455 1456 10 85.9 40431 258 1529 1530 1531 1532 10 85.9 40474 260 1537 1538 1539 1540 10 89.6 106742 352 1905 1906 1907 1908 10 93.2 143657 420 2177 2178 2179 2180 10 93.2 145800 434 2233 2234 2235 2236 10 96.4 9486 61 729 730 731 732 10 100.9 109666 383 2029 2030 2031 2032

EXAMPLE 2

This example illustrates a labeled probe degradation assay for SNP detection and marker mapping.

A quantity of maize genomic template DNA (e.g., about 2-20 ng) is mixed in 5 μLI total volume with four oligonucleotides, which can be designed by Applied Biosystems, i.e., a forward primer, a reverse primer, a hybridization probe having a VIC reporter attached to the 5′ end, and a hybridization probe having a FAM reporter attached to the 5′end as well as PCR reaction buffer containing the passive reference dye ROX. The PCR reaction is conducted for 35 cycles using a 60° C. annealing-extension temperature. Following the reaction, the fluorescence of each fluorophore as well as that of the passive reference is determined in a fluorimeter. The fluorescence value for each fluorophore is normalized to the fluorescence value of the passive reference. The normalized values are plotted against each other for each sample, producing an allelogram as described above. As described above, the data points should fall into clearly separable clusters.

To confirm that an assay produces accurate results, each new assay is performed on a number of replicates of samples of known genotypic identity representing each of the three possible genotypes, i.e., two homozygous alleles and a heterozygous sample. To be a valid and useful assay, it must produce clearly separable clusters of data points, such that one of the three genotypes can be assigned for at least 90% of the data points, and the assignment is observed to be correct for at least 98% of the data points. Subsequent to this validation step, the assay is applied to progeny of a cross between two highly inbred individuals to obtain segregation data, which are then used to calculate a genetic map position for the polymorphic locus.

The maize markers were genetically mapped based on the genotypes of certain SNPs. The genotypes were combined with genotypes for public core SSR and RFLP markers scored on recombinant inbred lines. Before mapping, any loci showing distorted segregation (P<0.01 for a Chi-square test of a 1:1 segregation ratio) were removed. These loci could be added to the map later but without allowing them to change marker order. A map was constructed using the JoinMap version 2.0 software, which is described by Stam (“Construction of integrated genetic linkage maps by means of a new computer package: JoinMap, The Plant Journal, 3: 739-744 (1993); Stam, P. and van Ooijen, J. W. “JoinMap version 2.0: Software for the calculation of genetic linkage maps (1995) CPRO-DLO, Wageningen). JoinMap implements a weighted-least squares approach to multipoint mapping in which information from all pairs of linked loci (adjacent or not) is incorporated. Linkage groups were formed using a LOD threshold of 5.0. The SSR and RFLP public markers were used to assign linkage groups to chromosomes. Linkage groups were merged within chromosomes before map construction.

Haldane's mapping function was used to convert recombination fractions to map distances. Lenient criteria was applied for excluding pairwise linkage data; only data with a LOD not greater than 0.001 or a recombination fraction not less than 0.499 are excluded. Parameters for ordering loci were a jump threshold of 5.0, a triplet threshold of 7.0 and a ripple value of 3. About 38% of the loci were ordered in two rounds of map construction with a jump threshold of 5.0, which prevents the addition of a locus to the map if such addition results in a jump of more than 5.0 to a goodness-of-fit criterion. The remaining loci were added to the map without application of such a jump threshold. Addition of these loci had a negligible effect on the map order and distances for the initial loci. Mapped SNP polymorphisms are identified in Table 2.

EXAMPLE 3

This example illustrates the utility of the markers in marker trait association

The 488 maize markers of this invention were used in an association study to identify which of the candidate genes were more significantly associated with oil level in corn (Zea mays).

The University of Illinois has corn lines differing in seed oil that have been developed by long-term selection. A high oil line (IHO) produces about 18% seed oil and a low oil line (ILO) produces about 1.5% seed oil. The IHO and ILO lines are available from the University of Illinois for research. A random mated population (RMn) was produced from random mating offspring of a cross between IHO and ILO by chain crossing for 10 generations to produce an RM10 population. From the RM10 population 504 S1-derived lines were developed by selling and these lines constitute an association study population. This population along with 72 control samples were genotyped using oil informative SNPs.

Phenotypes were measured on 504 association population lines in replicated field trials with an alph(0,1) incomplete block design. The field trials comprised the 504 lines grown in each of two years at each of 3 locations with 2 replicates per location. The lines were blocked within each replicate. These field trials were performed on the 504 RM10:S1 lines, per se, and on hybrids made by crossing each line to a tester line, i.e., line (7051).

Association was analyzed between the SNP markers and oil level in the RM10:S1 lines, per se, and in the hybrids. A mixed model analysis of variance was performed with sources of variation: location, reps within location, blocks and lines. Line effects estimated from this model were regressed on single marker genotypes (i.e., number of A alleles in the genotypes AA, Aa and aa). The probability that the slope is significantly different from zero gives an indication of whether the marker has a significant effect on the trait. Through this analysis of percent oil in the kernel and oil per 200 kernels in both inbreds and hybrids, a total of 186 markers showed significance at the p<0.05 level. These 186 significant markers are very likely to either reside within an oil gene or to be closely linked to an oil gene and arc more particularly described in Table 3 by:

“Map Position” which identifies the distance measured in cM from the 5′ end of a maize chromosome for the SNP identified by “Mutation ID”, which refers to an arbitrary identifying name for each polymorphism;

Pval % Oil Per se, which refers to probability of a test of significance of the regression of marker genotype on oil level as percent oil per kernel for inbred lines;

Pval % Oil Hybrid, which refers to probability of a test of significance of the regression of marker genotype on oil level as percent oil per kernel for hybrid lines.

Pval Oil/Kernel Per se, which refers to probability of a test of significance of the regression of marker genotype on oil level as oil weight per 200 kernels for inbred lines;

Pval Oil/Kernel Hybrid, which refers to probability of a test of significance of the regression of marker genotype on oil level as oil weight per 200 kernels for hybrid lines

TABLE 3 Pval Pval Pval Pval Map % Oil % Oil Oil/Kernel Oil/Kernel Position Mutation_ID Per se Hybrid Per se Hybrid 1-3.7 111829 0.706 0.234 0.336 0.046 1-25.1 43230 0.030 0.228 0.042 0.037 1-44 104827 0.094 0.801 0.018 0.909 1-45 151360 0.025 0.811 0.005 0.395 1-46.8 37716 0.009 0.113 0.024 0.351 1-53.3 42173 0.020 0.050 0.024 0.907 1-58.4 116 0.059 0.018 0.018 0.395 1-60.3 143100 0.722 0.029 0.878 0.501 1-60.6 33819 0.200 0.039 0.043 0.640 1-60.6 40189 0.007 1.6E−4 0.062 0.172 1-83.2 34205 0.026 0.151 0.090 0.022 1-86.3 8984 0.405 8.0E−4 0.433 0.069 1-86.3 36286 0.261 7.3E−4 0.328 0.069 1-88.8 29829 0.063 0.164 0.597 0.029 1-88.8 37068 0.026 0.317 0.068 0.051 1-90.5 111828 0.052 0.198 0.018 0.014 1-91 113263 0.281 0.004 0.078 0.489 1-91.8 104474 0.047 0.346 0.776 0.069 1-96.9 36448 0.006 0.114 0.002 0.052 1-99 40655 0.029 0.272 0.052 0.080 1-99 107077 9.7E−6 0.014 9.1E−4 0.021 1-103.3 8719 0.167 0.728 0.008 0.271 1-124.6 33373 0.029 0.240 0.201 0.714 1-130.3 69565 0.032 0.201 0.568 0.962 1-165.6 108862 0.011 0.001 0.402 0.347 1-178.6 151382 0.027 0.480 0.116 0.509 1-200.3 30840 0.662 0.050 0.716 0.012 2-5.8 31064 0.091 0.002 0.143 0.064 2-12.9 104447 0.077 0.012 0.697 0.459 2-14.1 39289 0.095 0.016 0.778 0.571 2-17.5 106678 0.048 0.003 0.043 0.040 2-19.5 82235 0.018 0.002 0.045 0.009 2-33.9 80031 0.101 0.046 0.557 0.036 2-35.9 13691 0.225 0.469 0.040 0.419 2-78.2 11466 0.096 0.761 0.045 0.225 2-78.2 79073 0.020 0.825 0.015 0.413 2-78.2 108493 0.142 0.045 0.713 0.299 2-92.5 3177 0.082 0.334 0.038 0.224 2-92.9 84829 0.298 0.324 0.111 0.031 2-99.7 151288 0.549 0.036 0.245 0.846 2-106 111475 0.238 0.013 0.320 0.685 2-106.2 108013 0.574 0.033 0.441 0.591 2-107.6 2307 0.497 0.019 0.437 0.413 2-114.9 22775 0.036 0.064 0.424 0.160 2-123.4 104954 0.049 0.058 0.573 0.765 2-152.4 43579 0.064 0.123 0.037 0.659 2-164.2 735 0.497 0.920 0.048 0.729 2-164.2 76792 0.939 0.524 0.044 0.345 3-6 8911 0.067 0.561 0.045 0.979 3-6 51614 0.071 0.551 0.030 0.980 3-9.1 10667 0.009 0.193 0.068 0.262 3-19.7 19963 0.115 0.084 0.029 0.373 3-19.7 32137 2.4E−4 1.1E−4 2.3E−4 0.037 3-46.2 49293 0.036 0.003 0.167 0.030 3-52.3 109315 0.175 7.7E−4 0.527 0.040 3-53.5 25000 0.098 4.5E−4 0.350 0.157 3-54.1 21154 0.060 7.7E−4 0.543 0.542 3-54.1 109722 0.482 0.022 0.526 0.284 3-57.2 109509 0.394 0.006 0.464 0.213 3-58.6 29867 0.036 1.8E−8 0.696 0.169 3-59.3 4599 0.093 7.9E−4 0.562 0.571 3-59.3 21190 0.020 0.006 0.637 0.215 3-59.3 28923 0.150 9.6E−4 0.703 0.351 3-59.3 147511 0.116 0.001 0.588 0.571 3-59.3 147768 0.066 7.6E−4 0.627 0.524 3-60.4 8685 0.592 0.001 0.913 0.681 3-61 16729 0.229 0.112 0.198 0.005 3-61.7 32247 0.115 3.5E−4 0.891 0.113 3-62.7 9144 0.066 0.003 0.277 0.014 3-62.7 9739 0.031 0.003 0.439 0.130 3-111.4 110780 0.246 0.040 0.572 0.207 3-123.8 143969 0.015 0.158 0.081 0.438 3-127.7 9079 0.040 0.071 0.296 0.134 4-38.7 110069 0.026 0.048 0.188 0.108 4-38.7 111464 0.029 0.053 0.129 0.096 4-52.8 24647 0.013 0.084 0.382 0.827 4-53.2 156243 0.004 0.007 0.096 0.368 4-62.1 10671 0.156 0.040 0.337 0.099 4-64.9 38852 0.285 0.072 0.342 0.007 4-69.5 5021 0.341 0.499 0.098 0.004 4-69.5 37503 0.262 0.126 0.303 0.002 4-69.9 107276 0.006 0.331 0.017 0.016 4-71.4 84527 0.346 0.014 0.363 0.040 4-80 106845 0.112 0.042 0.393 0.434 4-107.7 106491 0.020 0.040 0.409 0.521 4-112.4 54460 0.037 0.146 0.124 0.150 4-122.4 151472 0.186 0.994 0.011 0.967 4-128.1 32049 0.195 0.620 0.756 0.011 4-135.8 17900 4.2E−4 0.037 3.7E−4 0.019 4-136.4 147219 0.038 0.214 0.104 0.029 5-1.6 24265 0.082 0.035 0.472 0.010 5-39.9 109403 2.0E−6 9.1E−5 0.135 0.006 5-41.7 16527 0.028 0.161 0.333 0.791 5-50.9 109342 0.005 0.167 0.015 0.024 5-51.9 16762 7.6E−5 0.018 4.9E−4 0.029 5-51.9 16767 1.2E−4 0.017 5.1E−4 0.033 5-62.3 51419 0.046 0.002 0.163 0.031 5-63 32272 5.7E−5 0.001 0.008 0.100 5-66.9 30000 0.004 6.5E−4 0.035 0.002 5-66.9 146415 1.1E−4 1.9E−5 0.163 0.037 5-69.6 144731 8.5E−4 2.8E−4 0.162 0.042 5-70.5 105854 0.205 0.011 0.976 0.063 5-71.7 143216 0.023 0.014 0.065 0.098 5-76.4 29820 0.010 5.8E−4 0.128 0.140 5-80.2 36637 0.020 0.052 0.087 0.365 5-104.5 58375 0.028 0.097 0.024 0.003 5-150.5 31084 0.025 0.210 0.350 0.763 6-17.3 154854 0.010 0.222 0.049 0.688 6-30.8 69630 0.484 0.678 0.094 0.047 6-37.3 36067 0.018 0.290 0.215 0.874 6-37.3 36073 0.014 0.165 0.234 0.945 6-43.1 30176 0.827 0.323 0.969 0.015 6-52.8 4463 3.9E−9 1.8E−12 2.3E−6 3.7E−9 6-53.1 60751 3.9E−9 1.6E−6 5.4E−7 2.2E−4 6-53.5 32034 5.0E−7 3.7E−4 5.3E−5 0.048 6-53.5 57758 6.3E−5 0.008 0.002 0.566 6-53.5 108212 8.6E−7 6.0E−4 4.0E−5 0.043 6-58.1 59008 9.7E−5 3.9E−4 8.2E−5 0.002 6-58.1 146195 5.3E−4 8.5E−4 1.8E−5 0.004 6-59.9 3277 0.004 0.215 0.087 0.515 6-59.9 105586 0.003 4.9E−4 0.002 0.001 6-61.5 148039 0.120 7.6E−4 0.565 0.006 6-61.5 155861 0.082 7.2E−4 0.490 0.003 6-63.1 20410 0.028 0.012 0.055 0.138 6-66.6 8838 0.050 0.009 0.031 0.025 6-67.5 14694 0.226 8.2E−4 0.496 0.151 6-86.9 110972 0.023 0.050 0.072 0.482 6-110.4 31684 0.012 9.6E−4 0.162 0.240 6-121 37634 0.002 0.052 0.052 0.008 6-132.7 37555 0.089 0.364 0.665 0.025 7-62 42164 0.075 0.424 0.045 0.235 7-67 30674 0.424 0.048 0.187 0.015 7-68.7 39064 0.321 0.558 0.028 0.357 7-72.8 42930 0.111 0.076 0.006 0.002 7-74.2 68426 0.013 0.662 0.047 0.088 7-98.5 8799 0.031 0.429 0.009 0.160 7-98.8 48425 7.4E−4 0.063 2.6E−4 0.034 7-99.8 4415 6.9E−4 0.057 1.5E−4 0.032 7-99.8 35408 0.003 0.055 0.002 0.069 7-107.5 38914 0.024 0.002 0.682 0.747 7-115.8 4093 0.185 0.007 0.512 0.050 7-118.6 4302 0.032 6.5E−4 0.522 0.120 7-118.6 38653 0.199 0.011 0.471 0.035 7-118.6 81460 0.061 0.002 0.578 0.257 7-122.2 145260 0.062 0.003 0.108 0.003 7-124.5 15184 0.044 0.009 0.079 0.008 7-124.5 39773 0.065 0.022 0.814 0.608 7-132.8 30029 0.330 0.046 0.577 0.552 8-16.4 40320 0.657 0.063 0.405 0.006 8-40.9 107937 0.048 0.046 0.221 0.077 8-43.1 111628 0.105 0.011 0.401 0.144 8-45.5 26720 0.109 0.043 0.459 0.282 8-47.9 104862 0.152 0.143 0.011 0.276 8-53.9 27361 0.798 0.947 0.378 0.048 8-53.9 145200 0.260 0.129 0.033 0.016 8-55.7 23091 0.040 0.183 0.069 0.872 8-59.3 77568 0.003 0.258 4.4E−5 0.249 8-64 110148 0.005 0.085 0.005 0.528 8-65.8 104389 0.003 0.117 0.004 0.415 8-66.6 21895 0.003 0.143 0.002 0.417 8-67.4 48562 0.006 0.081 0.005 0.416 8-68.4 82295 0.003 0.311 4.9E−4 0.067 8-85.9 110684 0.039 0.588 0.030 0.082 8-87.5 9759 0.473 0.574 0.010 0.353 8-105.5 107286 0.028 0.063 0.104 0.076 8-106.8 13100 0.030 0.068 0.107 0.043 8-117.3 145077 0.006 0.009 0.055 0.192 8-117.3 145298 0.005 0.004 0.076 0.210 9-20.5 58904 0.578 0.351 0.078 0.041 9-80 29745 0.182 0.025 0.538 0.116 9-93.8 110377 0.021 0.097 0.019 0.134 9-93.8 113113 0.022 0.098 0.026 0.290 9-94 25961 0.058 0.047 0.039 0.177 9-94.5 148621 0.060 0.300 0.030 0.066 9-100.6 20048 0.034 0.137 0.155 0.156 9-100.6 153427 0.022 0.154 0.092 0.101 9-110.3 8937 0.014 0.652 0.035 0.233 9-125.2 9555 0.419 0.270 0.307 0.049 9-137.2 36022 0.136 0.014 0.290 0.096 10-52.7 143754 0.608 0.127 0.648 0.044 10-56.1 39275 0.061 0.019 0.054 0.085 10-89.6 106742 0.064 0.105 0.035 0.088 10-93.2 143657 0.560 0.046 0.790 0.186 10-93.2 145800 0.551 0.030 0.793 0.190 10-100.9 109666 0.083 0.007 0.144 0.148 unmapped 152577 0.315 0.001 0.627 0.914 unmapped 20742 0.425 0.141 0.435 0.041 

1. A method of identifying oil-associated QTLs in maize comprising (a) establishing a random mated population of maize plants derived from crossing the Illinois high oil line with the Illinois low oil line, (b) selecting a set of allelic polymorphic markers from the polymorphic genomic amplicon DNA sequences listed in Table 1, (c) identify in said set of allelic polymorphic makers a subset of oil informative markers having an allelic frequency difference of at least 0.6 between the Illinois high oil line and the Illinois low oil line, (d) identify kernel oil levels in individual plants of said random-mated population, (e) genotyping plants in said random-mated population using said subset of oil informative markers, (f) analyzing the association between said oil informative markers and said kernel oil levels to establish a statistical significance level, (g) select as oil QTLs genes those oil informative markers having a statistical significance of greater than 0.05.
 2. A method of claim 1 wherein said SNP markers are linked to markers mapped in Table
 2. 3. A method of claim 2 wherein the SNP markers are the markers listed in Table
 2. 4. A method of genotyping populations of maize plants comprising determining the presence in said plants of polymorphic allelic DNA for a marker listed in Table
 2. 5. A collection of sets of four oligonucleotides comprising a forward primer, a reverse primer, a FAM-labeled hybridization probe and a VIC-labeled hybridization probe for assaying a single SNP polymorphism identified in Table 2, wherein all of the oligonucleotides in said collection comprise nucleic acid molecules having DNA sequences of SEQ ID NO:485 through SEQ ID NO:2436.
 6. One set of four oligonucleotides from the collection of claim
 5. 7. A method of breeding maize comprising selecting a maize line having a polymorphism identified in Table 2 by SNP detection assay using a set of four oligonucleotides of claim
 6. 